Structural and Functional Studies on Cytochrome BO3 Ubiquinol Oxidase From Escherichia Coli and the Characterization of Its Quinone Binding Sites
Yap, Lai Lai
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https://hdl.handle.net/2142/84837
Description
Title
Structural and Functional Studies on Cytochrome BO3 Ubiquinol Oxidase From Escherichia Coli and the Characterization of Its Quinone Binding Sites
Author(s)
Yap, Lai Lai
Issue Date
2006
Doctoral Committee Chair(s)
Gennis, Robert B.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
Cytochrome bo3 ubiquinol oxidase is the terminal oxidase in the aerobic respiratory chain of Escherichia coli. The enzyme catalyzes the oxidation of ubiquinol-8 and the reduction of oxygen to water, which are coupled to the translocation of protons across the cytoplasmic membrane via protolytic scalar reactions as well as a proton pumping mechanism. A tightly but non-covalently bound ubiquinone is retained in the purified enzyme at the high affinity quinone binding (QH) site, and is able to transiently form a one-electron reduced semiquinone radical during the catalytic cycle. The semiquinone is highly stabilized at the QH site, and it was postulated from the crystal structure that ArgI71, AspI75, HisI98 and GlnI101 are the hydrogen bond partners involved in quinone binding in subunit I. Electron paramagnetic resonance (EPR) spectroscopic studies were carried out to characterize the semiquinone at the QH site of cytochrome bo3. Three exchangeable protons were identified around the semiquinone. The data supported a model in which the semiquinone exists as a protonated neutral radical, where ArgI71 and AspI75 were hydrogen bonded to the O1 carbonyl oxygen of the quinone ring to stabilize the semiquinone in a highly asymmetric manner. Two mutants, DI75E and D I75H, which alter the hydrogen bonding of the semiquinone, were also characterized. The presence of a second low-affinity quinone binding (Q L) site in cytochrome bo3 was demonstrated using the substrate analog Q1 and the inhibitors, 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) and aurachin C 1-10 (AC1-10). Solid-state nuclear magnetic resonance (SSNMR) studies of cytochrome bo3 were initiated, and spectra with high sensitivity and resolution were obtained. A cysteine-free mutant of cytochrome bo3 was constructed and cysteine residues were engineered in the vicinity of the entrances to the D-channel (RI134C) and K-channel (RmuI309C) for site-directed EPR spin labeling. The spectra obtained indicated no change in spin label motional characteristics upon cycling the enzyme through different oxidation states, thereby suggesting that there were no conformational changes associated with these redox changes. In summary, these studies have shed further insights into the structure-function relationship of cytochrome bo 3 and its elusive quinone binding sites.
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